Bio-polyphenols are super active substances, and can add a great deal to nutritional needs in health and disease. Their significant contribution towards health include antioxidative, antimicrobial, and possibly antidiabetic, anti-carcinogenic, and/or cardioprotective effects.
Cinnamomum spp, (cinnamon/cassia) contains essential oils, catechins, procyanidins, coumarins, tanning agents, phenolic carboxylic acids, lignans and mucins, in which coumarin is reported to be moderately toxic to liver and kidney (Giridhar Kanuri and Bergheim, Cinnamon and its medicinal effects, published online Apr. 23, 2009). Higher concentrations of coumarins can be measured particularly in certain types of cinnamon/cassia. Many extracts of cinnamon/cassia obtained in capsules were found to contain coumarins in high levels, (The Federal Institute for Risk Assessment, BfR Health Assessment No. 044/2006, 2006). As coumarins are generally soluble in alcohol, hydro-alcoholic extracts and alcoholic extracts may contain higher levels. The coumarin levels were found to be varying within various species of cinnamon and even different at barks collected from various parts within a tree ((Friederike W et. al., Journal of Agricultural and Food Chemistry, 2010, 58(19) pp. 10568-10575). This is a major concern in making use of the valuable cinnamon/cassia extracts as Nutraceutical/dietary supplement or food ingredient. Besides the high coumarin levels, the cinnamaldehyde levels in cinnamon powder capsules may possibly constitute a risk for pregnant women. Animal experiment studies indicate that taking cinnamaldehyde during pregnancy could lead to damage to the foetus (The Federal Institute for Risk Assessment, BfR Health Assessment No. 044/2006, 2006).
Recent research has mostly explored the benefits of cinnamon/cassia in blood-sugar management. In a study, Anderson et al (Journal of Agricultural and Food Chemistry, 2004, 52, pp. 65-70), isolated insulin action-enhancing complexes from the aqueous extracts of cassia/cinnamon and then characterized by nuclear magnetic resonance and mass spectroscopy as polymers composed of catechin/epicatechin with a molecular mass of 288, trimers with a molecular mass of 864 and tetramer with a mass of 1152. Their protonated molecular masses indicated that they are A type doubly linked procyanidin oligomers of the catechins and/or epicatechins. These polyphenolic polymers found in cinnamon/cassia may function as antioxidants, potentiate insulin action rendering cells more sensitive to insulin, and may be beneficial in the control of glucose intolerance and diabetes.
In another study an aqueous extract of cinnamon was reported to be effective in segregating the Alzheimer's disease causing proteins in vitro. (Peterson et al, Journal of Alzheimer's Disease, 2009, 17, 585).
In a recent study (Lu Z et. al, Phytomedicine, 2011, 18 (4), pp 298-302) the presence of both Type A and Type B procynidin polymers were established in various cinnamomum species and were shown to be bioactive.
Despite the health benefits of cinnamon/cassia polyphenols and procyanidins, no cost effective and commercially viable processing methods are available to selectively isolate coumarin free active polyphenols containing definite percentage of procyanidins, in sufficient purity, for nutraceutical, cosmeceutical and functional food ingredient applications.
Though the prior arts, U.S. Pat. No. 6,200,569 B1, US 2006/0013361 A1, US 2006/0013903 A1, and US 2009/7504118 B2 have detailed the isolation and various uses of cinnamon/cassia extracts containing procyanidins, such as for reduction of blood glucose and/or glycosylated hemoglobin levels, as a weight loss ingredient by reducing body fat and/or as a blood glucose lowering ingredient, to enhance creatine transport to muscles and for oral administration to control the blood glucose as well as improving glucose tolerance respectively, all these patents describe a highly temperature-time sensitive aqueous extraction protocol involving 50 to 200 times water. Though the method has been successfully applied to 1 to 50 g raw material, its commercial scale applicability has been seriously limited by various factors of the process such as the requirement of huge quantity of water and subsequent handling issues imparted by the mucilaginousin cinnamon/cassia bark, time-temperature sensitivity of the process and subsequent gel filtration using expensive adsorbents like Sephadex LH-20 and final concentration to powder. In addition no information has been given about the procyanidin content, polyphenol content, coumarin content, and organoleptic properties of the resulting extracts.
In another art US 2006/0073220 A1, an adsorbent resin based purification process for polyphenols and proanthocyanidins from various fruits and plant materials including cinnamon, using polystyrene copolymers containing electron withdrawing groups like bromine and non-substituted resins were detailed. Though high purity isolation of polyphenols and procyanidin polymers were claimed, no information has been given with respect to the polyphenol recovery from cinnamon, toxic and allergic coumarin and cinnamldehyde levels in the isolated polyphenols, and relative percentages of type A & B polymers that can be isolated from cinnamomum species. Though it is a general method of isolation of polyphenols from various natural raw materials, no informations have been given in the context of polyphenols or procyanidin type A & B polymers from Cinnamomum spp and their effect upon bioactivity.
In another US patent 2006 0275515 A1, a method of preparation of a cinnamon extract with antiviral properties against enveloped viruses including influenza A, parainfluenza and HSV-1 were mentioned. However no informations regarding the active molecules or procyanidin molecules or polyphenol content has been mentioned.
In another prior art US 2007/0292540/A1, the invention was fractionation of various chemical constituents in cinnamon. Supercritical carbon dioxide extraction of aroma fraction of cinnamon followed by aqueous extraction of the residue to obtain a polyphenolic acid and polysaccharide rich fraction were reported. The polyphenolic acid fraction was then passed through Sephadex LH 20 adsorbent resin to separate tannin phenolics from non-tannin phenolics. The disadvantage of this method is that it does not specify coumarin levels in various fractions, and does not provide any quantification and characterization of procyanidin levels in the final extract. Another limitation of this method is that it employs relatively expensive supercritical extraction method for the removal of the aroma compounds and expensive Sephadex LH 20 for the purification of polyphenols.
In a recent art US 2008/0306141 A1, procyanidin A-type polymer enriched extract was isolated from cinnamon bark using an organic solvent like acetone for extraction. Though this method can be used for bulk extraction, the percentage purity of the final product such as polyphenol content, or procyanidin Type A content etc is not assured. Another limitation of this method is that there is no plausible elimination of toxic coumarins from the procyanidin extract. It is also not possible to recover the aroma fraction of cinnamon bark, a valuable food flavourant. The method also does not confirm the purity of the procyanidins or the identity of the product as type A procyanidins. The prior art only mentions that the analysis was based on HPLC measurements.
In another prior art US 2010 0029756 A1, a method for the selective isolation of procyanidin type A tetramer with a molecular formula of C60H48O24 has been mentioned and its plausible applications as anti-inflammatory, anti microbial and bactericidal uses were speculated. The invention employed expensive instrumental techniques like multilevel preparative chromatographic instruments for the isolation and purification of the molecule. Further, no attempt have been mentioned to establish the level of bioactivity of the said molecule.
In another prior art US 2011/0039923 A1, the invention describes a process for the isolation of pentameric procyanidin rich cinnamon extract for antiviral applications. The disadvantage of the prior art is that it does not mention about the recovery of bioactive Type B polymers from cinnamon. Moreover, no information about the efficacy in relation with polyphenolic/procyanidin content has been furnished. In the present invention we describe a process for the isolation of total procyanidins including Type A and Type B polymers by eliminating the toxic coumarins and cinnamaldehyde.
The present invention attempts to overcome the hurdles in all these prior arts. In the present method, an easily scalable protocol is used to process cinnamon/cassia powder of the order of tones, using a 3 to 5 fold excess of water-based solvent for the isolation of polyphenols without coumarins.
An advantage of the present invention is an extraction method in which there is complete fractionation of cinnamon bark into a flavour ingredient and polyphenol powder can be achieved. The polyphenol powder was shown to be more than 90% pure (gallic acid equivalent) with procyanidins type A & B polymers, in which type A is present in 5 to 20% and type B polymer in 0.5 to 5%. Type A polymer was shown to be a mixture of doubly linked catechinlepicatechin trimer, tetramer, pentamer whereas Type B is a dimer formed by the B-type links.
Another advantage of the present invention is that it helps to selectively eliminate the toxic coumarins naturally present in many cinnamomum species and also helps to eliminate the other toxic or allergen components such as cinnamaldehye which is associated with the oil of cinnamon bark.
The present invention also delineate the level of polyphenol/procyanidin content with the bioactivity, as exemplified by the blood sugar levels, lipid levels and liver function markers. It is found that the bioactivity is directly proportional to the polyphenol/procyanidin content of the extract. As the polyphenol content increases from 15 to 75%, the blood sugar management potential was found to have a three-fold increase and the insulin potency increases 2.6 fold. The study also proved that coumarin and cinnamaldehyde contribution towards bioactivity is negligible as compared to the polyphenol content.
This invention can also be used for the direct extraction of polyphenols from cinnamon bark powder using water or alcohol or acetone or mixtures thereof and purification employing adsorbent chromatography.
The present invention also has the advantage that it can be easily scaled up to process tones of raw materials cost effectively. The invention also provides a unique method for the production of lower oligomers of polyphenols which are water soluble and more bioavailable.
The present invention also provides total isolation of polyphenols from cinnamon bark with an yield corresponding to greater than 90% recovery.
Another advantage of the method is to provide a process for the production of polyphenol isolates with high antioxidative index, ORAC value (Oxygen Radical Absorbing Capacity), not less than 3000 μmol Trolox equvalent/g.
Another advantage of the process is to derive a bioactive coumarin-free polyphenols in water soluble powder form as proved by the efficacy to control the blood sugar levels in streptozotocin induced diabetic rats.
Another advantage of the process is to optimize the polyphenol levels and procyanidin Type A & B levels for maximum bioactivity, as shown by the blood-sugar management study in streptozotocin induced diabetic rats
Another advantage of the present study is to provide safe and non toxic coumarin free bioactive polyphenols capable of reducing the LDL and, triglyceride levels in diabetic rats.
Another advantage of the present study is to provide a cinnamon bark extracted coumarin free polyphenols for maintaining the liver health of diabetic rats as monitored by the liver function markers alanine transaminase (ALT) and alakaline phosphatase (ALP).
Yet another advantage is the stable, directly compressible and non hygroscopic form of the polyphenolic extract for easy handling and formulation in tablet and capsule forms